As the change of ecological environment, people contacts with more and more sensitizing substance, which causes the incidence of allergic disease increasing. At present, approximately 25% of the population in the world is affected by allergic disorder. In 1998, detection of allergen and specific immunotherapy, announced by WHO, is the only etiological treatment which may affect the natural course of hypersensitivity disease and prevent the occurrence of new allergic diseases.
Specific immunotherapy treatment comprises identifying the species of allergen in a patient prior to making the patient contact with preparations containing this species of allergen repeatedly during non-acute stage, and the dosage increases gradually up to the best maintenance dosage so as to enhance the resistance of patients against said species of allergen to control or lessen the symptoms of hypersensitivity. The allergen preparation used refers to a biological product useful for diagnosing, prophylaxis and treatment of hypersensitivity disease. Said allergen preparation is obtained by extracting allergen from containing allergen ingredient natural materials (including insects, mites, fungi, animal danders, pollens, foods, etc), and then preparing a solution having a certain concentration of allergen, or further modifying the preparation by adsorbent or adjuvant.
In the past decades, professionals, organizations and agencies in Europe and America tried to develop an effective method for measuring allergen preparation. Though lots of methods were proposed, none of them had been accepted due to defects. For example, gravimetric volume (W/V) was used to represent the concentration of allergen extract. That is, a certain amount of degreased material (for example 1 g pollen which is degreased) was added into a certain amount (for example, 100 ml) of extraction fluid, then the W/V thereof was 1:100. Although it was very simple, the difference in factors, such as quality of the pollen collected and extraction procedure, may cause the ingredient, content and titer of the product having equivalent W/V value differently. In other words, W/V unit only indicates the concentration of extract, and it has no inevitable relationship with the titer or potency thereof. Additionally, protein nitrogen unit (PNU) was used to normalize the protein content in allergen extract because the main active component in allergen was protein. However, not all the proteins have antigenicity; and there is no definite relationship between the content of protein which has antigenicity and titer (i.e. the gross potency of all the allergic protein), so this method still could not be used to evaluate the gross potency of allergen product accurately. Therefore, WHO considered vaccine product normalized by the above two units as non-standardized product, because the allergen titer can not be compared between products from different manufacturers or different batches.
Biological unit (BU) and bioequivalent allergy unit (BAU) used respectively in Europe and America both are indications reflecting gross potency titer of allergen preparation, which is normalized by the result of direct skin test performed on hypersensitive patients. The BU in Europe was determined based on the dosage of allergen extract which causes the same stripe as histamine dichloride of certain concentration in a pricking test performed on 20 moderately hypersensitive patients, with the test results being represented by geometric mean. BAU was one important reference criteria for producing allergen preparation in US. Intracutaneous test was performed on 15 highly hypersensitive patients, and the titer of allergen was determined based on the arithmetic mean value, among all patients, of the concentrations which cause the patient to have 50 mm erythema in diameter. Both BU and BAU were based on in vivo allergic reaction in patients. That is, detection of IgE antibody in vivo against allergen may evaluate the gross titer more accurately. However, such methods depend on the availability of allergic patients as well as the criteria for selecting patients.
Methods for determining IgE in vitro to detect gross allergen potency include radioallergosorbent inhibitory test (RAST), enzyme linked immunosorbent inhibitory assay (ELISA), which are used to detect major allergen. These methods include radioallergosorbent inhibitory test (RAST-I) and many new in vitro assays developed based on RAST-I, such as MAST, FAST as well as Pharma-CAP, etc. Results of assay depend on the composition of serum pool and allergen extract bound to solid phase carrier, both of which are not reproducible. However, it is still impossible for such methods to standardize allergen preparation, because of the absence of stable serum pool between different batches.
Since allergen preparation is obtained by extraction from natural material, those factors, such as time, place and method for collecting, growth environment and extracting process of raw material, will increase the diversity of the quality of raw material. Moreover, allergens themselves are a complicated mixture of variants, and their epitopes are not entirely identical. Furthermore, immune reaction of individual patient is not the same. At present, methods for determining allergen potency in vitro all involve standardized serum, however, there is great difference between the serum collected each time. Therefore, it is difficult to control the quality of allergen preparation. The key to success of immune therapy lies in the use of high-quality allergen preparation which may be standardized and produced continuously. The purpose for standardization is to reduce the difference in product qualification and quantification, to increase safety, validity, reliability and accuracy, thus improving the diagnosis and treatment of allergic disease. The potency of allergen preparation is an important index for its quality control, since allergen preparation is used in diagnosis and treatment only after determining the potency of the preparation. However, it is impossible to compare products with each other, since the units used to indicate the results are not consistent when determining the potency of allergen.
Therefore, it is urgently desired in the art to provide a standardized serum mixture for detecting the potency of allergen preparation.